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Normal-phase liquid chromatography of plant hormones using reversed cholic acid micelles as the mobile phase.

When several plant hormones with similar structures are present in a sample, conventional analytical methods are not sufficiently selective to effectively differentiate them. However, the excellent selectivity of micellar liquid chromatography allows similarly structured compounds to be easily differentiated. We used reversed cholic acid micelles dissolved in tetrahydrofuran as the mobile phase in normal-phase chromatography of seven plant hormones. Our results conformed closely to those predicted by the pseudophase micellar liquid chromatography theory. The chromatographic efficiencies were calculated from the peak width and the retention times, and we compared these values with those obtained by reversed-phase chromatography using sodium dodecyl sulfate mobile phase in water. To test the efficiency of this selective method for the analysis of plant tissues that may also contain contaminants, we applied it to homogeneous suspensions of spiked maize roots. Recoveries ranged from 91 to 110%, the relative standard deviations were between 0.46 and 10.03%, and the detection limits were between 0.08 and 0.80 micrograms g-1.

Cholic Acids↗

[Separation and determination of eight plant hormones by reversed-phase high performance liquid chromatography].

In this paper, reversed-phase high performance liquid chromatographic technique was used for the separation and determination of eight plant hormones. Methanol-water-acetic acid system was chosen as the mobile phase. The effects of different separation conditions, such as the methanol and acetic acid concentrations in mobile phase, on the retention behaviours of eight plant hormones in this system were studied. The general trends in retention behaviours could be correlated to the methanol concentration in mobile phase. The experimental results showed that the optimum separation was achieved with following gradient elution condition: 0-3 minutes, 70% (water percentage in mobile phase), 3-13 minutes, 70%-20%, 13-48 minutes, 20%. Benzene was added to be as the internal standard. Under this experimental condition, the eight plant hormones could be separated completely and detected quantitatively at 260 nm within 16 minutes. The calibration curves for the eight compounds gave linearity over a wide range. The correlation coefficients of each components were r(ZT) = 0.9971, r(GAs) = 0.9999, r(K) = 0.9997, r(BA) = 0.9995, r(IAA) = 0.9998, r(IPA) = 0.9982, r(IBA) = 0.9995 and r(NAA) = 0.9995. The method is rapid, simple and efficient. It is a suitable method for the accurate determination of gibberellic acid (GA) and alpha-naphthaleneacetic acid (alpha-NAA) in products for agricultural use.

Chromatography, High Pressure Liquid↗

The plant hormone indoleacetic acid induces invasive growth in Saccharomyces cerevisiae.

Fungi must recognize plant-specific signals to initiate subsequent morphogenetic events such as filamentation that lead to infection. Here we show that the plant hormone indoleacetic acid (IAA) induces adhesion and filamentation of Saccharomyces cerevisiae. Genome expression profiling of cells treated with IAA identified Yap1, a fungal specific transcription factor, as a key mediator of this response. Strains lacking YAP1 (yap1-1) are hypersensitive to growth on IAA because they accumulate more IAA than can wild type. Members of a family of transporters the amino acid/auxin:proton symport permeases with homology to AUX1, a putative IAA transporter from plants, are up-regulated in the yap1-1 mutant. Deletion of any one of these transporters makes yap1-1 mutants more resistant to IAA by decreasing its uptake. The permease mutants are defective in IAA perception and filamentation. The ability of a fungus to perceive a plant hormone that causes it to differentiate into an invasive form has important implications for plant-pathogen interactions.

Dose-Response Relationship, Drug↗

Analysis of plant hormones in tobacco flowers by micellar electrokinetic capillary chromatography coupled with on-line large volume sample stacking.

Micellar electrokinetic capillary chromatography was developed to analyze plant hormones including gibberellic acid, abscisic acid, indole-3-acetic acid, alpha-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin-6-furfurylaminopurine and N6-benzyladenine. The influences of some crucial parameters including buffer concentration, pH value, micelle concentration and applied voltage on electrophoretic separation were investigated. Under optimum conditions (50 mM borate as the running buffer containing 50 mM sodium dodecylsulfate, pH 8.0; separation voltage: -15 kV; injection: hydrodynamic injection, 5 s at 50 mbar; temperature: 25 degrees C), a complete separation of seven plant hormones was accomplished within 30 min. Emphasis was placed on improving detection sensitivity in order to detect small amounts of hormones in plant tissue. Multiple wavelength detection and expanded bubble cell capillary were used with enrichment factors of 2 and 3, respectively. In addition, an on-line concentration method of large volume sample stacking was designed. Enrichment factors of up to approximately 10-600 were achieved for these hormones with detection limits down to 0.306 ng/ml. The method was successfully applied to analyzing abscisic acid in flowers of transgenic tobacco.

Buffers↗

The ethylene pathway: a paradigm for plant hormone signaling and interaction.

To dissect the web of signals that control plant growth, it is important to understand how the individual components of the pathway are modulated. Ethylene is a plant hormone involved in a large number of developmental processes. Biochemical and genetic approaches have provided a detailed view of the biosynthetic and signal transduction pathways of this hormone in the reference plant Arabidopsis thaliana. The effects of several hormones and of developmental changes on the regulation of the key enzymes of ethylene biosynthesis, ACC synthase and ACC oxidase, serve as a clear example of interaction between signals in the generation of complex responses. We now have a picture of how ethylene is sensed by the ethylene receptors and how the signal is further transduced to the nucleus. Although some of the ethylene receptors show a tissue-specific pattern of expression, little is known about the regulation of the components of the ethylene transduction cascade by other hormones or developmental factors. Once the ethylene signal reaches the nucleus, it activates a transcriptional cascade that results in changes in the expression of a number of genes. We describe some of the results that suggest an interaction at the transcriptional level between ethylene, other hormones, and stress signals.

Animals↗

Membrane-directed effects of the plant hormones abscisic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid.

This study examines two ways plant hormones might influence membrane processes, effects on overall permeability and modifications of specific ion channels. Abscisic acid (ABA) and indole-3-acetic acid (IAA) greatly enhanced erythritol permeability in mixed egg lecithin bilayers. In single component dioleoylphosphatidylcholine bilayers ABA was less effective than IAA, while 2,4-dichlorophenoxyacetate (2,4-D) did not affect either system or alter their ABA response. In Myxicola axons ABA and IAA had no effect, while 2,4-D (10 uM) caused a depolarizing shift of voltage-dependent Na+ and K+ activation by 25 +/- 4 mV and 15 +/- 3 mV, consistent with internal negative surface charge changes of -0.002 e-/A2 and -0.0007 e-/A2. We conclude that both generalized and ion channel-directed effects may link plant hormones and intracellular regulation.

2,4-Dichlorophenoxyacetic Acid↗

Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has an ability for production of scopoletin. In this cell culture, scopoletin is taken up from culture medium and accumulated in vacuoles after conversion to scopolin when cells are treated with 2,4-dichlorophenoxyacetic acid (2,4-D) (Taguchi et al. (2000)). To clarify the effect of 2,4-D on tobacco cells, its interaction with several other plant hormones was investigated. Other auxins also stimulated the uptake in the same manner as 2,4-D did, although higher concentrations were required than that of 2,4-D. When p-chlorophenoxyisobutyric acid (PCIB), an antiauxin, was added to the cell culture before 2,4-D, it inhibited 2,4-D-stimulated scopoletin uptake. This result suggests that the stimulation of scopoletin uptake was one of the auxin effects on tobacco cells. Among other classes of plant hormones that were tested, only salicylic acid stimulated the uptake. When these hormones were added to the cell cultures before 2,4-D, methyl jasmonate and kinetin reduced scopoletin uptake. These results suggest that this scopoletin uptake by tobacco cells is regulated by the interaction between different plant hormones.

Journal Article↗

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds.

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.

Chromatography, High Pressure Liquid↗

Differential activity of peroxidase isozymes in response to wounding, gypsy moth, and plant hormones in northern red oak (Quercus rubra L.).

We measured total peroxidase activity and the activities of peroxidase isoforms in leaves of red oak (Quercus rubra L.) seedlings exposed to wounding and plant hormones in the greenhouse. Activity of specific peroxidase isoforms was induced differentially by gypsy moth wounding, mechanical wounding, and the wound-associated plant hormone jasmonic acid. Activity of one isoform was enhanced modestly by treatment with salicylate. A study of peroxidase activity in naturally occurring galls elicited on red oak leaves by 12 hymenopteran and dipteran insect species found 16 POD isoforms, 11 of which were differentially induced or suppressed in galls compared with leaves. In both studies, total peroxidase activity as measured spectrophotometrically was not clearly related to activity of these isoforms. These results indicate that red oak seedlings and trees may respond specifically to wounding, particular insects, and plant signals through changes in the activities of individual isozymes.

Animals↗

Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar.

Sugars have signaling roles in a wide variety of developmental processes in plants. To elucidate the regulatory components that constitute the glucose signaling network governing plant growth and development, we have isolated and characterized two Arabidopsis glucose insensitive mutants, gin5 and gin6, based on a glucose-induced developmental arrest during early seedling morphogenesis. The T-DNA-tagged gin6 mutant abrogates the glucose-induced expression of a putative transcription factor, ABI4, previously shown to be involved in seed-specific abscisic acid (ABA) responses. Thus, ABI4 might be a regulator involved in both glucose- and seed-specific ABA signaling. The characterization of the gin5 mutant, on the other hand, reveals that glucose-specific accumulation of ABA is essential for hexokinase-mediated glucose responses. Consistent with this result, we show that three ABA-deficient mutants (aba1-1, aba2-1, and aba3-2) are also glucose insensitive. Exogenous ABA can restore normal glucose responses in gin5 and aba mutants but not in gin6 plants. Surprisingly, only abi4 and abi5-1 but not other ABA-insensitive signaling mutants (abi1-1, abi2-1, and abi3-1) exhibit glucose insensitivity, indicating the involvement of a distinct ABA signaling pathway in glucose responses. These results provide the first direct evidence to support a novel and central role of ABA in plant glucose responses mediated through glucose regulation of both ABA levels by GIN5 and ABA signaling by GIN6/ABI4.

Abscisic Acid↗

Regulation of the biosynthesis of plant hormones by cytochrome P450s.

Cytochrome P450 monooxygenases are a large group of heme-containing enzymes, most of which catalyze hydroxylation reactions. Since the discovery of cytochrome P450 in plants, more than 500 forms have been found, and they appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites. In particular, cytochrome P450s are involved in the biosynthesis of plant hormones, and play important roles in the regulation of plant growth and development. Recent genetic and functional analyses of cytochrome P450s in plants have significantly improved our understanding of not only the biosynthetic pathways themselves, but also of plant development from the perspective of hormonal control of morphogenesis. This review summarizes the present status of research on cytochrome P450s' roles in regulating the biosynthesis of plant hormones.

Journal Article↗

Plant hormone receptors: perception is everything.

Despite the bewildering ability of higher plants to change their development with respect to the environment, there appear to be only a few hormones that function to organize growth and development. With the recent identification of three plant hormone receptors, the molecular identities of all the major plant receptors are now known. Some plant hormones such as cytokinins, ethylene, and brassinosteroids (BR) use well-characterized signaling modules such as those involving receptor kinases, but in the case of the ethylene and BR receptors, there appear to be additional functions aside from the hormone they perceive. Auxin and gibberellin perception require unique mechanisms where the receptors are components involved in ubiquitination-dependent proteolysis. With plant hormone receptors in hand, comparisons can now be made between plants and other kingdoms as to how hormones control growth and development.

Models, Biological↗

Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.

A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of buffer pH, the concentration of HDB and the voltage on the resolution were studied systematically. By this method, the contents of plant hormone in biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery.

2,4-Dichlorophenoxyacetic Acid↗

Plant hormones in war and peace. Science, industry, and government in the development of herbicides in 1940s America.

This essay describes the emergence of "hormone" herbicides from academic plant physiology research in America in the late 1930s and 1940s, attending especially to the role of interactions between university scientists, industrial concerns, and government (particularly agricultural) agencies. The importance of an intellectual shift among the physiologists to viewing hormones as plant toxins rather than growth stimulators, spurred by wartime events, is discussed. The essay concludes by exploring the postwar marketing of these hormones as agrichemicals and as lawn treatments for suburban consumers, placing these in the economic and ecological context of other contemporary developments in farming technique.

Agrochemicals↗

Isolation of stress-related genes of rubber particles and latex in fig tree (Ficus carica) and their expressions by abiotic stress or plant hormone treatments.

Two rubber particle protein genes and one latex gene in fig tree (Ficus carica) have been isolated and their expression following various abiotic stress treatments have been investigated. The two major proteins that are tightly associated with the catalytically active rubber particles have been sequenced to be peroxidase (POX) and trypsin inhibitor (TRI). A cDNA encoding a basic class I chitinase (CHI) has also been isolated from the fig tree latex. Wounding treatment strongly induced the expression of the three stress-related genes. Among the abiotic stresses investigated, drought treatment greatly induced the expression of POX, whereas the expression of CHI and TRI decreased after the same treatment. Cold treatment reduced slightly the transcript levels of the thee genes, and NaCl reduced marginally the expression of CHI. The expression of POX, CHI, and TRI was induced by jasmonic acid and abscisic acid, by jasmonic acid, and by salicylic acid, respectively. Different expression of the stress-related genes following various abiotic stress or plant hormone treatments suggests that a crosstalk exists between the signal transduction pathways elicited by abiotic stresses and hormones in plants. Our present results showing the expression of stress-related proteins on the surface of rubber particles and latex in F. carica also imply the possible role of rubber particles and latex in defense in rubber-producing plant species.

Base Sequence↗

Induction of differentiation of human myeloid leukemia cells by jasmonates, plant hormones.

Some regulators of plant growth and differentiation have been shown to induce the differentiation of several human myeloid leukemia cells, and might be effective as differentiation inducers to control acute myelogenous leukemia cells. In this study, the growth-inhibiting and differentiation-inducing effects of jasmonates on human myeloid leukemia cells were examined. Several myeloid leukemia cells were cultured with methyl jasmonate (MJ) and its derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphological changes, alpha-naphthyl acetate esterase activity and expression of differentiation-associated surface antigens. MJ induced both monocytic and granulocytic differentiation of HL-60 cells. MJ activated mitogen-activated protein kinase (MAPK) in the cells before causing myelomonocytic differentiation. MAPK activation was necessary for MJ-induced differentiation, since PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MJ. MJ also induced the differentiation of other human leukemia cell lines. Introduction of a double bond at the 4,5-position greatly enhanced the differentiation-inducing activity of MJ. MJ and its derivatives potently induce the differentiation of some myelomonocytic leukemia cells. One novel derivative is a particularly promising therapeutic agent for the treatment of leukemia.

Cell Differentiation↗

The plant hormone auxin: insight in sight.

Physiological experiments conducted over the last 60 years indicate that the plant hormone auxin regulates a diverse set of developmental processes via changes in cell division, cell elongation and cell differentiation. Recent studies using transgenic plants with altered auxin levels support these conclusions and promise to provide more detailed information on the role of auxin during plant development. Although it is possible that all auxin responses are mediated by the same primary biochemical events, the studies described in this review are more consistent with multiple modes of auxin action. The development of molecular and genetic approaches to the study of hormone action should resolve this issue. The accelerated rate of progress in this field suggests that real insight into the mechanism of auxin action may be forthcoming.

Agrobacterium tumefaciens↗